Host Cell Vimentin Restrains Toxoplasma gondii Invasion and Phosphorylation of Vimentin is Partially Regulated by Interaction with TgROP18

He, Cheng; Kong, Ling; Zhou, Lijuan; Xia, Jing; Wei, Haixia; Liu, Min; Peng, Hongjuan

Cos7 cells were grown in 6-well culture plates and infected with RH tachyzoites for 0, 1, 2, 6, 12, 24, 36, and 48 h, then lysed with a high salt, detergent buffer (20mM Tris-HCl, pH 7.4; 600mM NaCl; 0.5% Triton X-100; 0.1mM sodium orthovanadate; and protease inhibitors (Roche)), and the soluble fraction separated from the insoluble fraction by centrifugation at 12,000g for 10 min at 4°C [53] . Equal volumes of soluble and insoluble fractions were loaded for SDS-PAGE and vimentin was detected with mouse anti-vimentin monoclonal antibody by western blotting (WB). Beta-actin was also detected by WB using a monoclonal antibody as a loading control. Details of primary and secondary antibodies are provided in Supplementary Material. The intensity of vimentin bands with non-saturated exposure from three independent experiments was analyzed using Image J software, and the proportion of soluble vimentin to total vimentin was calculated.